Notes on Two New Records of Mycobiota in Japan, Kostermansinda Nana and Durella Macrospora

Bull. Natl. Mus. Nat. Sci., Ser. B, 46(3), pp. 95–100, August 21, 2020

Notes on Two New Records of Mycobiota in Japan, Kostermansinda nana and Durella macrospora

Tsuyoshi Hosoya1, * and Michiko Yano2

1 Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan 2 Graduate School of Environment and Information Sciences, Yokohama National University, 79–7 Tokiwadai, Hodogaya, Yokohama 240–8501, Japan *E-mail: [email protected]

(Received 17 April 2020; accepted 24 June 2020)

Abstract Two new records of mycobiota in Japan were documented, i.e. Kostermansinda nana and Durella macrospora. While the latter clearly belongs to Helotiales, the phylogenetic position of the former was not clear based on BLAST search on ITS-5.8S, LSU, mtSSU, RPB1, RPB2, and EF1a.

Key words: barcode, Durella macrospora, Japan, Kostermansinda nana, mycobiota, taxonomy.

Introduction diversity Research, National Museum of Nature In spite of continuous attempts in elucidation and Science. Coordinates followed WGS84 of mycobiota in Japan, a number of taxa have datum. been remained unreported. In the present paper, Extraction of DNA, PCR and sequencing fol- we documented two new records of fungi lowed Iatagaki et al. (2019). The following scarcely known. To increase the value of their primer sets were used. For internal transcribed occurrence data, we also added genetic informa- spacers (ITS1 and ITS2) and 5.8S ribosomal tion for the future phylogenetic analysis. region (ITS-5.8S), ITS1 and ITS4 (for Koster- mansinda nana) or ITS1Fand ITS4 (for Durella macrospora) (White et al., 1990); for large sub- Materials and Methods unit of ribosomal RNA, LR0R and LR5 (Vilgalys For the isolation of Kostermansinda nana, sin- and Hester, 1990); for mitochondrial small sub- gle spore isolation method using Skerman’s unit of ribosomal RNA, mrSSU1 and mrSSU3R manipulator (Skerman, 1968) was used. Isolate (Zoller et al., 1999); for DNA-directed RNA of Durella macrospora was obtained from multi- polymerase II subunit RPB1, RPB1-AF and spores discharged onto the agar as previously RPB1-CR (Castlebury et al., 2004); for partial described (Itagaki et al., 2019). Isolates were sequence of the second largest DNA-directed deposited in Biological Resource Center, RNA polymerase II subunit (RPB2), RPB2-P6F National Institute of Technology and Evaluation and RPB2-P7R (Liu et al., 1999); for partial (NITE-BRC, Tokyo, Japan, https://www.nite.go. sequence of elongation factor 1 (EF1a), EF1- jp/en/nbrc/cultures/index.html). Extracted DNA 983F and EF2218R (Rehner and Buckley, 2005). were deposited to the Center for Molecular Bio- BLAST search was conducted based on the obtained sequences using the NCBI website © 2020 National Museum of Nature and Science (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO 96 Tsuyoshi Hosoya and Michiko Yano

GRAM=blastn&BLAST_PROGRAMS=mega walled, multi-septate, brown, forming synnemata. Blast&PAGE_TYPE=BlastSearch&SHOW_ Synnemata upright, 200–300×20–40 μm, com- DEFAULTS=on&LINK_LOC=blasthome, visited posed of dark-brown, multi-septate, smooth, on March 15, 2020). Only sequences matched unbranched hyphae of 2–3 μm wide. Conidio- with E＝0.0 and higher percent identity value phores macronematous, thick-walled. Conidio- were selected for discussion. genous cells monoblastic, integrated, terminal, determinate. Conidia 20–30×12–15 μm, soli- tary, acrogenous, broadly ellipsoidal clavate, Results and Discussion muriform, typically composed of three rows and Descriptions 3–5 levels of cells with pale to dark-brown wall, 1. Kostermansinda nana (J.N.Kapoor & Munjal) arranged on one smaller hyaline basal cell. Ger- G.Z.Zhao [as ‘nanum’], Journal of Shandong mination occurs while the conidia are still on the Agricultural University 34(3): 437. 2003. conidiophores, and detached conidia germinate [Fig. 1] readily on artificial agar media. ≡Sclerographium nanum J.N.Kapoor & Munjal, Specimens examined. TNS-F-24817 (culture Indian Phytopath. 19(4): 352. 1967 [1966]. FC-1894＝NBRC 112551), 24818, and 24819 on ＝Kostermansinda minima Cabello & Aramb., in bark of unidentified tree, Myoriki-ji, Iryuda, Arambarri, Cabello & Mengascini, Mycotaxon Odawara-shi, Kanagawa Pref. 2009-III-20. col. 29: 32. 1987. T. Hosoya; TNS-F-25226 and 25227 on unidenti- Mycelia immersed in the substrate, thick- fied log, Takizawa-en, Tanzawa, Kanagawa Pref.

Fig. 1. Kostermansinda nana (TNS-F-24817). A. Synnemata. B. Spores detached and scattered on the substrate. C. Synnnemata connected with other synnema at the basal hyphae. D. Close up of the head apical region of the synnema. E. Close up of the head of synnema with a cluster of conidia. F. Crush mount of the conidia pro- ducing part. Note that the conidia are produced at the end of brown hyphae. G. Conidia and conidiogenous cells with terminal enlargement. H. Detached conidia, with globular, hyaline cell at the base. I. Juvenile conidia and end of conidiogenous cells. J. Germinating conidium on agar mediuma (potato dextrose agar). Scales: A–B＝100 μm, C–I＝10 μm, J＝20 μm Kostermansinda nana and Durella macrospora 97

(35.398717, 139.183162), Oct., 1989. col. T. and Heleiosa (Eriomycetaceae, order unknown) Hosoya; TNS-F-37038 (culture FC-2752 were listed in this order. ＝NBRC 109679) on bark of living tree, Jinmuji, For mtSSU, Anisomeridium sp. (Monoblastia- Zushi-shi, Kanagawa Pref., (33.43705, ceae, Monoblastiales, Dothideomycetes) isolate 132.637803), 2010–X–11. col. M. Yano. e4012523987 (MH028639; Max score 710, Sequences registered. LC534830 (ITS-5.8S), Query coverage 97%, and %Ident. 83.78%), fol- LC534831 (LSU), LC535799 (mtSSU), lowed by Megalotremis verrucosa (Monoblastia- LC535800 (RPB1), LC535801 (RPB2), and ceae) (GU327694; 710, 86%, and 85.80%), LC535802 (EF1α) obtained from NBRC 112551 Trypetheliopsis kalbi (Monoblastiaceae) Notes. Kostermansinda is a relatively newly (GU327703; 652, 86%, and 84.27%) were listed described genus, containing small number of in the range of E＝0.0. species. Kostermansinda nana has been collected For RPB1, no matches were obtained with several times in Japan, and previously known as E＝0.0. K. minima, but no description was provided. The For RPB2, Wiesneriomyces javanicus fungus has been known from Argentina (Aram- (Wiesneriomycetaceae, Tubeufiales) isolate YMF barri et al., 1987), India (Bhat, 2006), Taiwan 1.04036 (MK267285; 726, 94%, and 84.01%), (Chang, 1989) and Thailand (Kodsueb et al., W. javanicus isolate YMF 1.04044 (MK267286; 2008), and this is the first occurrence report of 721, 94%, and 83.82%) were obtained. Kostermansinda in Japan. For EF1α, many matches were obtained, but The phylogenetic position of Kostermansinda no match was presented for more than has remained unresolved, and it is disposed in ＞90%Ident value. The highest match showed “Ascomycota” in Index Fungroum (http://www. Dictyocheirospora (Dictyosporiaceae, Pleospora- indexfungorum.org/names/Names.asp, visited on les). March 10, 2020), and inc. sedis in Wijayawar- The above results are a bit confusing, but dene et al. (2020). In the present study, BLAST allow preliminary consideration about the posi- search based on several genes are presented as tion of this mysterious fungus. Some of the follows (higher taxonomy followed Wijayawar- BLAST search result suggest the phylogenetic dene et al. 2020). relationship of K. nana with Dothideomycetes. When ITS-5.8S was used for BLAST search, This suggestion is also supported by brown to the only genus specified with E＝0.0 was Entro- black, dictyosporous conidia often observed in phospora infrequnentan (Glomeromycota) this class. NY349 (U94714) with Max score 662, Query Another possible interpretation brings up a coverage 96%, and %Ident. 93.89% which is too doubt for the correct identification of the regis- low to evaluate taxonomic position based on the tered sequences. BLAST search listed some similarity. lichen members (members of Lecanorales). LSU provided more genera as candidates in These sequences may have been originated from E＝0.0, but no genus was specified for %Ident lichenicolous Dothideomycetes. The latter possi- ＞90%. Below %Ident ＜90%, Funbolia (Erio- bility can be eliminated by incorporating multi- mycetaceae, order unknown), Phellinocrescentia ple sequences resulted from multiple specimens (Eriomycetaceae, order unknown), Tucker- or examination of voucher specimen. However, manopsis (listed as orthologous variant Tucker- due to the paucity of the genomic data for allied mannopsis; Parmeliaceae, Lecanorales), Hypo- taxa, further analysis was not conducted. gymnia (Parmeliaceae, Lecanorales), Satchmopsis Cochlearomycetaceae, Leotiales?), 2. Durella macrospora Fuckel, Jb. nassau. Ver. Pleurosticta (Parmeliaceae, Lecanorales), Bryo- Naturk. 23–24: 281. 1870 [1869–70]. ria (Parmeliaceae), Bryocaulon (Parmeliaceae), [Figs. 2, 3] 98 Tsuyoshi Hosoya and Michiko Yano

Fig. 2. Durella macrospora (TNS-F-61703). A–B. Dried specimens. C. Surface of ectal excipulum in crush mount. Note upper portion composed of undulating hyphae and lower portion composed of larger, thicker- walled cells. D. Brown epitheical structure. E. Ascus, F. Ascospore. G. Paraphyses apex, H. Ascal apex, I. Asci and undulating paraphyses in crush mount. Scales: A＝1 mm, B＝0.5 mm, C＝20 μm, D–I＝10 μm.

Apothecia superficial, gregarious, sessile, tur- Specimen examined. TNS-F-61703 (culture binate to shallowly concave, totally black when FC-5679＝NBRC 114601), on unidentified wood, fresh, drying shallow to flat, attached to the Renkoji, Tama-shi, Tokyo (35.636111, 139.4600), neighboring apothecia, 120–250 μm in diameter. 2015-II-12. col. M. Nakajima. Ectal excipulum textura intricata, composed of Sequences registered. LC534832 (ITS-5.8S) undulating hyphae with elongate cells with Notes. This is the first occurrence report of brown-colored wall with thin, fewer septa Durella in Japan. It is previously known mainly 2–4 μm wide; wall becoming thicker toward the from Europe and America but so far no records margin ending up in somewhat clavate terminal from Asia (GBIF, https://www.gbif.org/, visited cell; cells shorter, thicker-walled and more pris- on April 25, 2020). Overall morphology agrees matic (10–25×5–8 μm, with 1 μm thick wall) with Durella connivens (Fr.) Rehm (Hansen and toward the base. Asci 60–88×7–9 μm, cylindri- Knudsen, 2000; Medardi, 2004; Luis et al., cal clavate, arising from croziers; apex rounded; 2017), but differs in the morphology of paraphy- pore stained faintly in Melzer’s reagent without ses and MLZ positive asci, and agrees with mor- 3% KOH pretreatment, greatly enhanced by 3% phology of D. macrospora (Medardi, 2004). The KOH pretreatment. Ascospores 10–19×3–4 μm BLAST search based on ITS-5.8S region shows (13.47±2.24×3.37±0.44 μm in average±sd, almost perfect match with two sequences for D. n＝15), biseriate or biseriate only in upper macrospora [D. macrospora voucher G.M. 2015- region; clavate or ellipsoid, often narrower 04-05-4 (KY462813; Max score 1064, Query toward the pointed base, aseptate to three-sep- coverage 100%, and %Ident. 99.32%) and D. tate. Paraphyses filiform, 1.5–2 μm wide at the macrospora voucher H.B. 9974 (KY462812; base, undulate, simple or branched at the base or 1064, 100%, 99.32%)]. When considering the apical region, thicker-walled and enlarged toward 98.5% identify species limit (Nilsson et al., the apex, up to 5 μm wide, brown-colored at the 2012). The present specimen matches well with apex; almost the same length or slightly exceed- the previously reported sequences, and shows no ing the asci, exudating brown, resinous matter doubt for species identity on molecular basis. forming epithecium. However, some contradiction with the previous Japanese name. Ko-kuro-sara-byotake description given by Dennis (1956) are noted. Kostermansinda nana and Durella macrospora 99

branched, and more swollen in Dennis (1956) as compared with the present specimen (Fig. 3D). Nevertheless, we identiﬁed this species based on the high similarity of the barcoding region. Johnston et al. (2019) included D. macrospora in their phylogenetic analysis of Leotiomycetes, and demonstated its close relationship with Arachnopezizaceae. Baral (2016) placed the genus Durella in Strossmayeria lineage, next to Mollisiaceae, but Baral (2016) also pointed out the morphological diﬀerences between Mollisiaceae. We also agree with the morphological similarity of Durella in Strossmayeria, in particular ectal excipular structure, ascospores and paraphyses.

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